Use of zfp36 as a target in anti-non-small cell lung cancer drugs and pharmaceutical compositions

Abstract

This invention discloses the application of ZFP36 as a target in anti-non-small cell lung cancer (NSCLC) drugs and pharmaceutical compositions, belonging to the field of biomedical technology. The invention upregulates ZFP36 expression or enhances its RNA-binding activity, utilizing the zinc finger domain composed of amino acids 115-148 of the ZFP36 protein to specifically bind to the ATTTA-type ARE motif of the FASN mRNA 3'UTR and promote target mRNA degradation, thereby downregulating the expression of FASN, lipid droplet-related proteins, and CD36, thus triple-inhibiting lipid droplet metabolism. Simultaneously, it inhibits CAF secretion of lipid carriers, blocking lipid transport and metabolic remodeling in the tumor microenvironment. This invention can inhibit NSCLC cell proliferation, induce apoptosis, and reverse chemotherapy resistance. It clarifies that ZFP36-related biomarkers can be used for lung cancer chemotherapy resistance subtyping and targeted efficacy prediction, providing a novel target and approach for precision treatment of NSCLC, and possessing excellent clinical translational value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ZFP36 as a target in anti-non-small cell lung cancer drugs and pharmaceutical compositions. Background Technology

[0002] Non-small cell lung cancer (NSCLC) accounts for more than 85% of all lung cancer cases and is the leading cause of cancer-related deaths worldwide, with a 5-year overall survival rate of less than 20% for patients with intermediate or advanced stages. Platinum-based combination chemotherapy is the first-line standard treatment for intermediate and advanced NSCLC, but the incidence of chemotherapy resistance exceeds 60% in clinical practice, which is a core factor leading to treatment failure and shortened patient survival. Currently, there is no effective targeted therapy to reverse chemotherapy resistance.

[0003] Recent studies have confirmed that abnormal lipid droplet accumulation is one of the core metabolic characteristics of malignant tumor cells. Lipid droplets not only provide energy and biomembrane synthesis raw materials for the rapid proliferation of tumor cells, but also mediate chemotherapy resistance in tumor cells by scavenging intracellular reactive oxygen species and isolating chemotherapeutic drugs. Tumor-associated fibroblasts (CAFs) in the tumor microenvironment can secrete lipid carriers to provide exogenous lipid sources for tumor cells, further exacerbating the abnormal accumulation of lipid droplets and chemotherapy resistance phenotype within tumor cells. Therefore, simultaneously targeting tumor cell-mediated autonomous lipid droplet synthesis and intercellular lipid transport in the tumor microenvironment is a key breakthrough for reversing chemotherapy resistance in NSCLC and improving treatment efficacy.

[0004] ZFP36 is a classic zinc finger RNA-binding protein. Its core function is to bind to the AU enrichment element (ARE motif) of the 3' untranslated region (3'UTR) of target mRNA through its zinc finger domain, thereby promoting the degradation of target mRNA and participating in various physiological and pathological processes such as inflammation regulation, lipid metabolism, and tumorigenesis and development. Although existing studies have reported the downregulation of ZFP36 expression in lung cancer tissues, its role and mechanism in lipid metabolism remodeling in NSCLC have not been systematically elucidated. In particular, no studies have confirmed that ZFP36 can simultaneously regulate intracellular lipid droplet biosynthesis and CAF-mediated intercellular lipid transport in tumor cells. Furthermore, there is no mature technical approach based on ZFP36 to regulate lipid droplet accumulation for the treatment of NSCLC and to reverse chemotherapy resistance. Therefore, it cannot provide an effective target and drug development direction for the precise targeted therapy of NSCLC. Summary of the Invention

[0005] The purpose of this invention is to provide an application of ZFP36 as a target in anti-non-small cell lung cancer drugs and a pharmaceutical composition. By upregulating ZFP36 expression or enhancing its RNA binding activity, the zinc finger domain composed of amino acids 115-148 specifically binds to the ATTTA motif of FASN mRNA 3'UTR and promotes its degradation, downregulating the expression of FASN, lipid droplet-related proteins, and CD36, thus triplely inhibiting lipid droplet metabolism. At the same time, it inhibits CAF secretion of lipid carriers, blocks lipid transport in the tumor microenvironment, reconstructs lipid metabolism, and ultimately inhibits lung cancer cell proliferation, induces apoptosis, and reverses chemotherapy resistance. Furthermore, the ZFP36 expression level and the ZFP36 / FASN / CD36 ratio can serve as molecular markers for lung cancer chemotherapy resistance classification and prediction of targeted efficacy.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The application of ZFP36 as a target in anti-non-small cell lung cancer drugs involves upregulating ZFP36 expression or enhancing its RNA binding activity. On the one hand, it inhibits the biosynthesis and abnormal accumulation of lipid droplets in non-small cell lung cancer tumor cells. On the other hand, it inhibits the secretion of lipid carriers by tumor-associated fibroblasts (CAFs) and blocks intercellular lipid transport in the tumor microenvironment. This, in turn, regulates the lipid metabolism remodeling of the tumor microenvironment and exerts an anti-non-small cell lung cancer effect.

[0007] Furthermore, ZFP36 specifically binds to two core AU enrichment elements (ARE motifs) in the 3' untranslated region (3'UTR) of fatty acid synthase (FASN) mRNA through the zinc finger domain composed of amino acid residues 115-148 of the ZFP36 protein shown in SEQ ID NO:2. The core sequence of the ARE motif is ATTTA, thereby promoting the degradation of FASN mRNA. Through the above-mentioned regulatory effects, ZFP36 can simultaneously downregulate the expression levels of FASN protein, lipid droplet-related proteins Perilipin-2 and PLIN3, and lipid droplet transporter CD36, achieving triple inhibition of lipid droplet biosynthesis, abnormal accumulation, and intercellular transport.

[0008] Furthermore, knocking down the expression of ZFP36 can prolong the half-life of FASN mRNA by ≥2.5 times; the C124R and C150R mutants of the ZFP36 zinc finger domain completely lose their ability to bind to FASN mRNA and cannot exert the regulatory effect described in claim 2.

[0009] Furthermore, the non-small cell lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, and large cell neuroendocrine carcinoma, preferably non-small cell lung cancer resistant to platinum-based, taxane-based, antimetabolite, or anthracycline chemotherapy drugs; the chemotherapy-resistant non-small cell lung cancer cells include A549R, PC9R, H1975R, and H460R cells; the chemotherapy drugs include at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed, and doxorubicin.

[0010] Furthermore, the ZFP36 expression level is a specific molecular marker for distinguishing between chemotherapy-resistant and chemotherapy-sensitive non-small cell lung cancer; by detecting the expression levels of ZFP36, FASN, and CD36 in the sample, the ratio of ZFP36 to FASN and CD36 expression levels is used to predict the efficacy of targeted therapy for non-small cell lung cancer.

[0011] A pharmaceutical composition for treating non-small cell lung cancer, comprising an active ingredient and a pharmaceutically acceptable carrier, excipient, or solubilizer; said active ingredient is a ZFP36 expression upregulator or a ZFP36 RNA binding activity enhancer.

[0012] Furthermore, the ZFP36 expression upregulator is selected from at least one of the following: (1) Gene therapy vectors carrying the ZFP36 coding sequence or its functionally active fragments; (2) ZFP36 small molecule agonist, wherein the small molecule agonist, when treated with non-small cell lung cancer A549 cells at a concentration of 10 μM for 72 h, upregulated ZFP36 expression by ≥3.5 times, reduced lipid droplet accumulation by ≥55%, downregulated FASN protein expression by ≥45%, and downregulated CD36 expression by ≥40%; (3) Demethylating drugs, histone deacetylase inhibitors, or epigenetic activators targeting the ZFP36 promoter region; (4) Nucleic acid aptamers, microRNA inhibitors, lncRNA antagonists or circular RNA agonists that enhance the transcription or translation efficiency of ZFP36; (5) A recombinant fusion protein that retains the ZFP36 zinc finger domain, wherein the recombinant fusion protein can bind to both FASN mRNA and CD36 promoter region.

[0013] Furthermore, the gene therapy vector is selected from AAV2 / 6 adeno-associated virus vectors, tumor-targeting lentiviral vectors, lipid nanoparticle (LNP) vectors, or tumor cell-derived exosome vectors; the vector contains the ZFP36 coding sequence shown in SEQ ID NO:1 or its functionally active fragment; the functionally active fragment is a zinc finger domain coding fragment that retains the ability to bind the FASN mRNA 3'UTRARE motif, and can be stably expressed in non-small cell lung cancer cells and CAFs; the vector can be modified with tumor-targeting peptides to improve the targeted delivery efficiency of tumor tissues and CAFs and reduce off-target toxicity.

[0014] Furthermore, the active ingredient also includes a chemotherapy drug or a lipid metabolism targeting inhibitor; the chemotherapy drug is selected from at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and pemetrexed; the lipid metabolism targeting inhibitor is selected from at least one of FASN-specific inhibitors, ACC inhibitors, AMPK activators, and CD36 inhibitors.

[0015] Furthermore, the dosage form of the pharmaceutical composition is a nebulized inhaler, an intratumoral injection, an intravenous injection, an oral preparation, a pleural infusion, or a sustained-release implant.

[0016] The proposed scheme is based on the dual and synergistic regulatory role of the RNA-binding protein ZFP36 in both autonomous lipid metabolism in tumor cells and lipid transport in the tumor microenvironment. Through post-transcriptional sequence-specific gene expression regulation, it achieves full-chain blockade of lipid droplet metabolism in non-small cell lung cancer, ultimately exerting biological effects of inhibiting tumor progression and reversing chemotherapy resistance. The specific mechanism is as follows: The ZFP36 protein, through its characteristic zinc finger domain consisting of amino acid residues 115-148 in the sequence shown in SEQ ID NO:2, recognizes and binds to two AU-enriched elements (ARE motifs) with the core sequence ATTTA in the 3' untranslated region of fatty acid synthase (FASN) mRNA in a strictly sequence-specific manner, directly initiating the degradation process of the target mRNA. This binding and regulation is absolutely structure-dependent; when the zinc finger domain undergoes loss-of-function mutations such as C124R or C150R, ZFP36 completely loses its ability to bind to FASN mRNA, and its downstream regulatory effects also disappear completely. Through the aforementioned post-transcriptional regulation, ZFP36 significantly downregulates the protein expression of FASN, a key rate-limiting enzyme in fatty acid synthesis, and simultaneously inhibits the expression of lipid droplet-related proteins Perilipin-2 and PLIN3, thereby blocking the biosynthesis and abnormal accumulation of endogenous lipid droplets in tumor cells at the source. Furthermore, it directly inhibits the expression of the lipid droplet transporter CD36, blocking the intracellular uptake of exogenous lipids, thus achieving a triple inhibition of lipid droplet biosynthesis, abnormal accumulation, and intercellular transport. Simultaneously, ZFP36 can inhibit the lipid carrier secretion function of tumor-associated fibroblasts (CAFs), blocking lipid transport and nutrient supply from CAFs to tumor cells in the tumor microenvironment, disrupting the lipid metabolism interaction between tumor cells and stromal cells, reconstructing the abnormal lipid metabolism pattern of the tumor microenvironment, and eliminating the supporting role of the tumor microenvironment for the malignant phenotype of tumor cells.

[0017] Compared with the prior art, the beneficial effects of the present invention are: Existing technologies mostly target only the endogenous lipid droplet synthesis in tumor cells, generally neglecting the driving role of tumor-associated fibroblast (CAF)-mediated intercellular lipid transport in the tumor microenvironment on tumor progression and drug resistance. Furthermore, the core regulatory role of ZFP36 in lipid metabolism remodeling in non-small cell lung cancer (NSCLC) has not been systematically elucidated. This invention is the first to demonstrate that ZFP36 can specifically bind to FASN mRNA through its zinc finger domain and promote its degradation, simultaneously achieving a triple inhibition of lipid droplet biosynthesis, abnormal accumulation, and intercellular transport in tumor cells. It also blocks the lipid metabolism interaction between CAF and tumor cells, fundamentally breaking down the abnormal lipid metabolism phenotype of NSCLC, providing a novel target and mechanistic pathway for targeted therapy of NSCLC.

[0018] In current clinical practice, the incidence of chemotherapy resistance in NSCLC exceeds 60%, which is the core reason for treatment failure, and there is a lack of safe and effective reversal methods. This invention, by upregulating ZFP36 expression or enhancing its activity, can effectively reverse the resistance phenotype of first-line clinical chemotherapy drugs such as platinum-based, taxane-based, antimetabolites, and anthracyclines. When ZFP36 expression upregulators are used in combination with chemotherapy drugs, the cell viability of chemotherapy-resistant NSCLC can be reduced by ≥85%, the tumor inhibition rate in vivo can be increased to over 90%, and the cardiotoxicity and nephrotoxicity of chemotherapy drugs can be significantly reduced. This not only greatly improves the treatment effect of drug-resistant NSCLC but also reduces the adverse reactions of chemotherapy, solving a long-standing clinical treatment dilemma.

[0019] Current technologies lack chemotherapy resistance classification and efficacy prediction biomarkers that match lipid metabolism-targeted therapy, making precise patient stratification impossible and easily leading to ineffective treatment. This invention clarifies that ZFP36 expression level is a specific molecular marker for distinguishing between chemotherapy-resistant and sensitive phenotypes in NSCLC, and that the expression ratio of ZFP36 / FASN / CD36 can accurately predict the efficacy of targeted therapy. This provides clinicians with a standardized tool for patient stratification, applicable population screening, and efficacy prediction, effectively avoiding ineffective chemotherapy, reducing patient treatment costs and adverse reaction risks, and truly achieving individualized precision treatment for NSCLC. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the core mechanism of action of the ZFP36 target against non-small cell lung cancer in this invention. Figure 2 This is a diagram showing the composition and dosage form architecture of the drug composition for treating non-small cell lung cancer according to the present invention. Detailed Implementation

[0021] The technical solutions of the present invention will now be described in detail with reference to the accompanying drawings.

[0022] To achieve the above-mentioned objectives, this invention provides the following core technical solutions through systematic in vitro and in vivo experiments and clinical sample verification: The first core technical solution: Application of substances that upregulate ZFP36 expression or enhance its RNA binding activity in the preparation of drugs for treating non-small cell lung cancer. The substance, by upregulating ZFP36 expression or enhancing its RNA binding activity, inhibits the biosynthesis and abnormal accumulation of lipid droplets in non-small cell lung cancer tumor cells on the one hand, and inhibits the secretion of lipid carriers by tumor-associated fibroblasts (CAFs) and blocks intercellular lipid transport in the tumor microenvironment on the other hand, thereby regulating the lipid metabolism remodeling of the tumor microenvironment and achieving the effect of anti-non-small cell lung cancer.

[0023] Furthermore, ZFP36 specifically binds to two core AU enrichment elements (ARE motifs) in the 3' untranslated region (3'UTR) of fatty acid synthase (FASN) mRNA through the zinc finger domain composed of amino acid residues 115-148 of the ZFP36 protein shown in SEQ ID NO:2. The core sequence of the ARE motif is ATTTA, thereby promoting the degradation of FASN mRNA. Through the above-mentioned regulatory effects, ZFP36 can simultaneously downregulate the expression levels of FASN protein, lipid droplet-related proteins Perilipin-2 and PLIN3, and lipid droplet transporter CD36, achieving triple inhibition of lipid droplet biosynthesis, abnormal accumulation, and intercellular transport.

[0024] Furthermore, knocking down ZFP36 expression can prolong the half-life of FASN mRNA by ≥2.5 times; the C124R and C150R mutants of the ZFP36 zinc finger domain completely lose their ability to bind to FASN mRNA and cannot exert the above-mentioned regulatory effects.

[0025] Further, the non-small cell lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, and large cell neuroendocrine carcinoma, preferably non-small cell lung cancer resistant to platinum-based, taxane-based, antimetabolite, or anthracycline chemotherapy drugs; the chemotherapy-resistant non-small cell lung cancer cells include A549R, PC9R, H1975R, and H460R cells; the chemotherapy drugs include at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed, and doxorubicin.

[0026] Furthermore, the drug is used to inhibit the proliferation of non-small cell lung cancer cells, induce tumor cell apoptosis and senescence, enhance tumor cell DNA damage, arrest the G1 / S phase cell cycle of tumor cells, improve the chemotherapy sensitivity of non-small cell lung cancer, and prolong the progression-free survival of non-small cell lung cancer subjects.

[0027] Furthermore, ZFP36 expression level can serve as a specific molecular marker to distinguish between chemotherapy-resistant and chemotherapy-sensitive non-small cell lung cancer, and the expression ratio of ZFP36 / FASN / CD36 can predict the efficacy of targeted therapy for non-small cell lung cancer.

[0028] The second core technology solution: a drug composition for treating non-small cell lung cancer. The pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable carrier, excipient, or solubilizer; the active ingredient is a ZFP36 expression upregulator and / or a ZFP36 RNA binding activity enhancer; the pharmaceutical composition exerts its anti-non-small cell lung cancer effect by inhibiting lipid droplet accumulation in tumor cells, downregulating the expression of FASN and lipid droplet-related proteins, and blocking CAF-mediated lipid droplet transport.

[0029] Furthermore, the ZFP36 expression upregulator is selected from at least one of the following: (1) Gene therapy vectors carrying the ZFP36 coding sequence or its functionally active fragments; (2) ZFP36 small molecule agonist, wherein the small molecule agonist, when treated with non-small cell lung cancer A549 cells at a concentration of 10 μM for 72 h, upregulated ZFP36 expression by ≥3.5 times, reduced lipid droplet accumulation by ≥55%, downregulated FASN protein expression by ≥45%, and downregulated CD36 expression by ≥40%; (3) Demethylating drugs, histone deacetylase inhibitors, or epigenetic activators targeting the ZFP36 promoter region; (4) Nucleic acid aptamers, microRNA inhibitors, lncRNA antagonists or circular RNA agonists that enhance the transcription or translation efficiency of ZFP36; (5) A recombinant fusion protein that retains the ZFP36 zinc finger domain, wherein the recombinant fusion protein can simultaneously bind to FASN mRNA and CD36 promoter region, thereby doubly inhibiting lipid droplet synthesis and transport.

[0030] Furthermore, the gene therapy vector is selected from AAV2 / 6 adeno-associated virus vectors, tumor-targeting lentiviral vectors, lipid nanoparticle (LNP) vectors, or tumor cell-derived exosome vectors; the vector contains the ZFP36 coding sequence shown in SEQ ID NO:1 or its functionally active fragment; the functionally active fragment is a zinc finger domain coding fragment that retains the ability to bind the FASNmRNA3'UTRARE motif, and can be stably expressed in non-small cell lung cancer cells and CAFs; the vector can be modified with tumor-targeting peptides to improve the targeted delivery efficiency of tumor tissues and CAFs and reduce off-target toxicity.

[0031] Furthermore, the active ingredient also includes chemotherapeutic drugs and / or lipid metabolism targeting inhibitors; the chemotherapeutic drugs are selected from at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and pemetrexed; the lipid metabolism targeting inhibitors are selected from at least one of FASN-specific inhibitors, ACC inhibitors, AMPK activators, and CD36 inhibitors. The ZFP36 expression upregulator has a synergistic antitumor effect with the chemotherapeutic drugs and lipid metabolism inhibitors, reducing the viability of chemotherapy-resistant non-small cell lung cancer cells by ≥85%, increasing the tumor inhibition rate to over 90% in a nude mouse xenograft tumor model, and simultaneously reducing the cardiotoxicity and nephrotoxicity of chemotherapeutic drugs.

[0032] Furthermore, the pharmaceutical composition can reverse the abnormal lipid metabolism phenotype of chemotherapy-resistant non-small cell lung cancer cells, restore their chemotherapy sensitivity, and has no significant cytotoxicity to normal lung tissue endothelial cells and epithelial cells.

[0033] Furthermore, the dosage form of the pharmaceutical composition is a nebulized inhaler, an intratumoral injection, an intravenous injection, an oral formulation, an intrapleural infusion, or a sustained-release implant; wherein the nebulized inhaler has a lung-targeted bioavailability of ≥70% calculated by the area under the drug concentration-time curve in rat lung tissue using LC-MS / MS; the intratumoral injection has a sustained-release period of ≥72 hours in vitro in pH 7.4 PBS buffer; and the sustained-release implant can achieve continuous release of ZFP36 expression upregulator, with the dosing cycle extended to once every 4 weeks.

[0034] This solution, based on the pharmaceutical composition of the present invention, provides a personalized dosing method adapted for clinical translation, specifically as follows: Patient stratification screening: By detecting the expression level of ZFP36, the ZFP36 / FASN / CD36 expression ratio, and the degree of lipid droplet accumulation in the patient's tumor tissue, patients suitable for ZFP36 targeted therapy are screened. Administration method and dosage: Based on the patient's tumor stage, pathological type and lesion location, select the appropriate drug dosage form and administration route, including nebulized inhalation, intratumoral injection, intravenous injection, intrapleural perfusion or sustained-release implantation, and formulate individualized dosing dosage and cycle; Combination therapy regimen: For chemotherapy-resistant patients, ZFP36 expression upregulators are combined with clinical standard chemotherapy drugs, while the dosage of chemotherapy drugs is reduced simultaneously, thereby improving the anti-tumor effect and reducing chemotherapy toxicity. Efficacy monitoring and regimen adjustment: Regularly monitor the expression levels of ZFP36, FASN, and Perilipin-2 in the patient's tumor tissue / peripheral blood, as well as changes in tumor volume, and dynamically adjust the dosage and cycle of medication based on the efficacy.

[0035] The preparation of AAV2 / 6 gene therapy vectors supplemented with the ZFP36 coding sequence includes the following steps: Total RNA was extracted from human normal lung endothelial cells, and cDNA was obtained by reverse transcription. The full-length coding sequence of human ZFP36 shown in SEQ ID NO:1 was amplified by PCR. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; and 72℃ final extension for 10 min. The PCR products were separated by 1% agarose gel electrophoresis, the target fragment was recovered by gel excision, and sequencing verified that the sequence matched SEQ ID NO:1 100%.

[0036] The amplified ZFP36 coding sequence was digested with XhoI / NotI and cloned into the pAAV-MCS expression vector to construct the pAAV-ZFP36 recombinant vector. At the same time, the pAAV-EGFP empty control vector was constructed. Enzyme digestion and sequencing verified that the vector was constructed correctly and that there were no base mutations or frameshifts.

[0037] The recombinant vector was co-transfected with AAV helper plasmids pHelper and pAAV-RC2 / 6 at a mass ratio of 1:1:1 into HEK293T cells with a confluence of 80%-90%. PEI was used as the transfection reagent. After transfection, the medium was replaced with fresh DMEM medium containing 10% fetal bovine serum 6 h later, and the cells were cultured at 37°C and 5% CO2 for 72 h.

[0038] After culture, scrape off cells, centrifuge at 3000 rpm for 10 min at 4°C to collect cell pellet, resuspend in PBS and freeze-thaw three times (flash freeze in liquid nitrogen for 3 min, thaw in a water bath at 37°C for 5 min), centrifuge at 12000 rpm for 30 min at 4°C to collect supernatant and remove cell debris.

[0039] AAV2 / 6-ZFP36 viral particles were purified using cesium chloride density gradient ultracentrifugation. The ultracentrifugation conditions were 4℃, 200,000g for 18h. Viral components were collected stepwise and dialyzed three times with PBS for 4h each time to remove cesium chloride impurities. Viral titers were detected by RT-qPCR with primer sequences of: forward 5'-GGAACCCCTAGTGATGGAGTT-3' and reverse 5'-CGGCCTCAGTGAGCGA-3'. Viral vectors with a titer ≥1×10¹² vg / mL were finally obtained. After passing sterility and endotoxin tests, the viral vectors were aliquoted into sterile cryovials and stored at -80℃ protected from light.

[0040] The AAV2 / 6-ZFP36 vector was modified with the tumor-targeting peptide RGD (sequence: Arg-Gly-Asp-Cys). The modification ratio was 1 mg of RGD peptide per 1 × 10¹² vg of viral particles. The vector was incubated at 4 °C for 12 h and then dialyzed to remove unbound target peptides, thereby improving the targeted delivery efficiency of tumor tissue and CAF and reducing off-target toxicity.

[0041] The preparation steps for a pharmaceutical composition containing a ZFP36 upregulator in combination with chemotherapy drugs are as follows: Basic formulation (100mL preparation): The active ingredients are the AAV2 / 6-ZFP36 carrier (final concentration 1×10¹¹ vg / mL) prepared in Example 1 and cisplatin (final concentration 1 mg / mL). Pharmaceutically acceptable excipients include: mannitol 5g (isotonic regulator), Tween-80 0.1g (suspending agent), appropriate amount of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH adjuster), and sterile physiological saline to a final volume of 100mL, with the pH adjusted to 7.2-7.4.

[0042] Preparation method: Under Class A aseptic conditions, mannitol and Tween-80 were first added to an appropriate amount of sterile physiological saline and stirred until completely dissolved. Phosphate buffer was added to adjust the pH to 7.3. Then, AAV2 / 6-ZFP36 carrier stock solution and cisplatin injection were added and stirred evenly. Sterilized solution was then brought to a final volume with sterile physiological saline. The solution was filtered through a 0.22μm sterile membrane terminal filter for sterilization, dispensed into 10mL sterile vials, capped, and stored at 4℃ protected from light. The shelf life is tentatively set at 24h.

[0043] Quality control: The drug is released after passing sterility testing, endotoxin testing (endotoxin content <0.25 EU / mL), virus titer testing, and cisplatin content testing. This drug composition can simultaneously exert the lipid droplet metabolism regulation effect mediated by ZFP36 and the cytotoxic effect of cisplatin. The two have a significant synergistic anti-tumor effect and can effectively reverse chemotherapy resistance in NSCLC.

[0044] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. These embodiments are merely descriptions of preferred embodiments and are not intended to limit the scope or concept of the invention. The specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. Such combinations, as long as they do not violate the spirit of the present invention, should also be considered as part of this disclosure. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations.

[0045] This invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this invention and without departing from the design idea of ​​this invention, all modifications and improvements made by those skilled in the art to the technical solutions of this invention should fall within the protection scope of this invention. The technical content for which protection is sought in this invention has been fully described in the claims.

Claims

1. The use of ZFP36 as a target point in an anti-non-small cell lung cancer drug, characterized in that, By up-regulating the expression of ZFP36 or enhancing its RNA binding activity, on the one hand, the biosynthesis and abnormal accumulation of lipid droplets in non-small cell lung cancer tumor cells are inhibited, on the other hand, tumor associated fibroblasts (hereinafter referred to as CAF) are inhibited to secrete lipid carriers, block the intercellular lipid transport in the tumor microenvironment, and then regulate the lipid metabolism reconstruction of the tumor microenvironment, thereby playing an anti-non-small cell lung cancer role.

2. Use according to claim 1, characterized in that, The ZFP36 specifically binds to two core AU-rich elements (hereinafter referred to as ARE motifs) of the fatty acid synthase (hereinafter referred to as FASN) mRNA 3' untranslated region (3'UTR) through the zinc finger domain composed of the 115-148 amino acid residues of the ZFP36 protein shown in SEQ ID NO: 2, and then promotes the degradation of the FASN mRNA; through the above regulation, ZFP36 can simultaneously down-regulate the expression levels of FASN protein, lipid droplet related proteins Perilipin-2, PLIN3, and lipid droplet transporter CD36, and achieve triple inhibition of lipid droplet biosynthesis, abnormal accumulation and intercellular transport.

3. Use according to claim 2, characterized in that, After the expression of the ZFP36 is knocked down, the half-life of the FASN mRNA is extended by ≥2.5 times; the C124R, C150R mutants of the ZFP36 zinc finger domain completely lose the binding ability to the FASN mRNA and cannot exert the regulation effect of claim 2.

4. Use according to any one of claims 1 to 3, characterized in that, The non-small cell lung cancer includes lung adenocarcinoma, lung squamous carcinoma, large cell neuroendocrine carcinoma, and preferably platinum, taxane, anti-metabolite or anthracycline chemotherapeutic drug resistant non-small cell lung cancer; the chemotherapeutic drug resistant non-small cell lung cancer cells include A549R, PC9R, H1975R, H460R cells; the chemotherapeutic drugs include at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed, and doxorubicin.

5. Use according to any one of claims 1 to 3, characterized in that, The expression level of ZFP36 is a specific molecular marker for distinguishing chemotherapeutic drug resistant and chemotherapeutic drug sensitive non-small cell lung cancer; by detecting the expression amounts of ZFP36, FASN and CD36 in a sample, the expression amount ratio of ZFP36 to FASN and CD36 is used to predict the efficacy of non-small cell lung cancer targeted therapy.

6. A pharmaceutical composition for the treatment of non-small cell lung cancer, characterized by comprising the compound of claim 1 as an active ingredient. The active ingredient is a ZFP36 expression up-regulator or a ZFP36 RNA binding activity enhancer.

7. The pharmaceutical composition of claim 6, wherein, The ZFP36 expression up-regulator is selected from at least one of the following: (1) a gene therapy vector carrying a ZFP36 coding sequence or a functionally active fragment thereof; (2) a ZFP36 small molecule agonist, which, when treated at a concentration of 10 μM for 72 h in non-small cell lung cancer A549 cells, makes the expression of ZFP36 up-regulated by ≥3.5 times, the accumulation of lipid droplets reduced by ≥55%, the expression of FASN protein down-regulated by ≥45%, and the expression of CD36 down-regulated by ≥40%; (3) a demethylation drug, a histone deacetylase inhibitor or an epigenetic activator targeting the promoter region of ZFP36; (4) Nucleic acid aptamers, microRNA inhibitors, lncRNA antagonists or circular RNA agonists that enhance the transcription or translation efficiency of ZFP36; (5) A recombinant fusion protein that retains the ZFP36 zinc finger domain, wherein the recombinant fusion protein can bind to both FASN mRNA and CD36 promoter region.

8. The pharmaceutical composition of claim 7, wherein, The gene therapy vector is selected from AAV2 / 6 adeno-associated virus vectors, tumor-targeting lentiviral vectors, lipid nanoparticle (LNP) vectors, or tumor cell-derived exosome vectors; the vector contains the ZFP36 coding sequence shown in SEQ ID NO:1 or its functionally active fragment; the functionally active fragment is a zinc finger domain coding fragment that retains the ability to bind the 3'UTRARE motif of FASN mRNA, and can be stably expressed in non-small cell lung cancer cells and CAFs; the vector can be modified with tumor-targeting peptides to improve the targeted delivery efficiency of tumor tissues and CAFs and reduce off-target toxicity.

9. The pharmaceutical composition of claim 6, wherein, The active ingredient also includes a chemotherapy drug or a lipid metabolism targeting inhibitor; the chemotherapy drug is selected from at least one of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and pemetrexed; the lipid metabolism targeting inhibitor is selected from at least one of FASN-specific inhibitors, ACC inhibitors, AMPK activators, and CD36 inhibitors.

10. The pharmaceutical composition of claim 6, wherein, The dosage form of the pharmaceutical composition is a nebulized inhaler, an intratumoral injection, an intravenous injection, an oral preparation, a pleural infusion, or a sustained-release implant.

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